The fundamental task of pathogen recognition receptors (PRRs) is to initiate an immediate inflammatory response against invading pathogens to quickly control infection; however, inappropriate or prolonged activation of PRRs leads to immunodeficiency, autoimmunity, and severe tissue damage. Since many members of the NLR (Nod-like receptor) and RLR (RIG-I-like receptor) families of PRRs have been implicated in autoinflammatory diseases, it will be especially important to examine the negative regulators of these proteins to understand the existing mechanisms for reversing PRR activation. A primary negative regulator of the RLR family of PRRs has been identified in our recent work demonstrating that the linear ubiquitination assembly complex (LUBAC) is a critical negative regulator of RIG-I (Inn KS, et al, Molecular Cell, In Press). We have shown that the HOIL-1L subunit of LUBAC blocks RIG-I activation by targeting a positive regulator of RIG-I for degradation by linear ubiquitination and by directly binding to RIG-I. Particularly, my preliminary data demonstrates that HOIL-1L also binds NLRP1 (NACHT LRR and PYD domain-containing protein 1), a member of the NLR family of PRRs, which oligomerizes upon ligand binding in the cytoplasm to form 'inflammasome' complexes to activate proinflammatory caspase 1 and cytokine IL- 1b. The interaction between LUBAC and members of both the RLR and NLR families of PRRs suggests that LUBAC is a master regulator of PRRs; however, the biological consequences of the interaction with NLRP1 are unknown. To address this gap in knowledge, I aim to test the hypothesis that LUBAC negatively regulates NLRP1- mediated inflammation by targeting NLRP1 for degradation via linear ubiquitination, which will be tested in three specific aims. In the first aim, I will determine whether NLRP1 is a substrate for ubiquitination by LUBAC. In the second aim, I will establish whether the HOIL-1L/NLRP1 interaction negatively regulates NLRP1-inflammasome induced signaling in cell culture by HOIL-1L overexpression, mutation, and knockdown. In the third aim, the expression of NLR family members and NLRP1-mediated inflammation will be examined in the HOIL-1L-/- mouse to determine the regulatory role of LUBAC in vivo. Additionally, the NLRP1 L115H allele strongly associated with vitiligo, type I diabetes, and Addison's autoimmune disease susceptibility will be assessed for HOIL-1L binding to determine significance of the NLRP1/HOIL-1L interaction for the molecular basis of human diseases. Collectively, the experiments in all three aims will examine the significance and consequences of the interaction between HOIL-1L and NLRP1 to address the gap in our understanding of the negative regulation of inflammation and provide new insight for the underlying molecular mechanisms of NLRP1-mediated diseases. Since NLRP1 is one of over 20 proteins in the NLR family of PRRs, which have been implicated in a wide range of autoinflammatory diseases, my work may more broadly address the regulation of other NLRs and inform the design of therapeutics that manipulate NLR-inflammasome activation to treat autoinflammatory disease.